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Comparative Genomics Final Results Ben Dan Deepak Esha Kelley Pramod Raghav Smruthy Vartika Will Questions to be Addressed 1. Sixteen strains clustered with V. navarrensis type strain LMG15976 • • • 16S rRNA, pyrH, recA and rpoA Four formed a distinct cluster V. vulnificus Closest relative to both lineages of V. navarrensis “Is it a different species or biotype?” 2. V. navarrensis strains isolated from various sources. • nav_2423 (VN1) : Blood • nav_2462 (VN2) : Surface Wound • nav_2541 (VN3) : Sewage • nav_2756 (VN4) : Water “Is Vibrio navarrensis pathogenic?” Genes common/unique to V.vulnificus and V.navarrensis SPECIATION?? Whole genome super matrix tree VN3 97.13 100% Bootstrap support >0.98 posterior probability support VN2 95.58 VN4 98.35 82.64 VN1 VV1 98.60 98.88 97.76 81.94 98.92 VV4 VV2 VV3 VV5 Vp1 0.02 Aligned using Clustal-omega. A concatenated alignment was generated and a bootstrapped (100) maximum likelihood phylogenetic tree was generated using the Jones-Taylor-Thornton model of evolution and an assumption of a constant rate of change. The tree was rooted with Vibrio parahaemolyticus as an outgroup. All nodes had 100% bootstrapping support and >0.98 posterior probability support (8 chains for 20,000 generations sampling at every 100th generation). ANI support for each node is shown. ANI values for internal nodes were calculated by taking the average ANI for all pairs of genomes representing the bifurcation. 16S rRNA Tree 12 VV1 VV2 VN4 VN2 11 Vibrio_vulnificus_CMCP6 Vibrio_vulnificus_YJ016 VN1 74 VV4 95 VV5 VN3 VV3 Vibrio_parahaemolyticus 0.015 0.010 0.005 0.000 • 16S is not informative for differentiating closely related Vibrio species. • Full length 16S rRNA sequences were assembled by mapping to the reference . • Aligned using PyNAST • Bootstrapped ML tree was generated using MEGA • Rooted using V. parahaemolyticus PATHOGENECITY?? Approach I Approach II Annotated Dataset Gene Predictions Reference Strains Annotation Files from NCBI Existence of Toxins Presence Absence Machinery for Incorporation Machinery for Incorporation Yes Correlation with Pathway (KEGG) Connecting the dots Pathogenic or Putatively Pathogenic OrthoMCL Potentially Pathogenic Generation of presence-absence matrix Test for group significance (ANOSIM test) No Unlikely Pathogenic ID genes associated with groups (SIMPER test) Heatmaps in R to view gene profiles Different combos of files Approach II (contd) Gene files Blast Parser Upload parsed data to Database PreProcessing All v/s All Blast Find Protein Pairs Filter Fasta BlastDb Markov Clustering Cluster of Orthologs Gene Profiles for All Strains V. fischeri 14 V. fischeri 11 V. splendidus 32 Non-human pathogens V. cholerae 61 V. cholerae 95 V. parahaemolyticus 33 V. parahaemolyticus 0P Pathogens V. vulnificus YJ016 V. vulnificus CMCP06 VV01 VV02 VV03 VV04 VV05 VN01 VN04 VN03 VN02 60 70 80 90 Similarity 100 Group Average dendrogram generated from a simple matching resemblance matrix. Presence Absence Gene Profiles for All Strains VV05 VV04 VN04 VN02 VV02 VN01 VV01 VN03 2D Stress: 0.09 VVYJ VVCM VC61 VC95 VV03 path vul nonpath nav VS32 VP33 VP0P VF14 VF11 MNDS plot generated from a simple matching resemblance matrix. The dendrogram is a bit misleading about the relationship between V. splendidus and V. fischeri. ANOSIM Statistical Test ANOSIM is a nonparametric method that tests whether two or more groups of samples are significantly different. R statistic - A measure of the strength of the difference between two groups. A value closer to +1 signifies more dissimilarity between the groups Significance Level - tests the significance of the difference. Analogous to p-value. Groups R statistic Significance Level % Pathogenic, V. vulnificus 0.487 0.6 Pathogenic, Non-Pathogenic 0.37 6 Pathogenic, V. navarrensis 0.712 1 V. vulnificus, Non-Pathogenic 1 1.8 V. vulnificus, V. navarrensis 1 0.8 Non-Pathogenic, V. navarrensis 1 2.9 Genes significantly different between V. vulnificus and V. navarrensis V. fischeri ES114 V. fischeri MJ11 V. spendidus LGP32 V. cholerae O1 biovarE1 Tor N16961 V. cholerae O395 V. parahaemolytics RIMD_2210633 V. parahaemolytics BB220P V. vulnificus YJ016 V. vulnificus CMCP6 VV01 VV02 VV03 VV04 VV05 VN01 VN04 VN03 VN02 60 70 80 90 Similarity 100 Presence Absence Genes significantly different between V. vulnificus and V. navarrensis V. fischeri ES114 V. fischeri MJ11 V. spendidus LGP32 V. cholerae O1 biovarE1 Tor N16961 V. cholerae O395 V. parahaemolytics RIMD_2210633 V. parahaemolytics BB220P V. vulnificus YJ016 V. vulnificus CMCP6 VV01 VV02 VV03 VV04 VV05 VN01 VN04 VN03 VN02 60 70 80 90 Similarity Presence Absence 100 Hypotheticals / Conserved hypotheticals Presence Absence Missing from V. navarrensis V. fischeri ES114 V. fischeri MJ11 V. spendidus LGP32 V. cholerae O1 biovarE1 Tor N16961 V. cholerae O395 V. parahaemolytics RIMD_2210633 V. parahaemolytics BB220P V. vulnificus YJ016 V. vulnificus CMCP6 VV01 VV02 VV03 VV04 VV05 VN01 VN04 VN03 VN02 60 70 80 90 Similarity 100 Unique to V. navarrensis n-acetyl transferase acetyl transferase glucokinase Heme Biosynthesis / Iron acquisition Adhesin Chemotaxis Genes significantly enriched in a priori defined “Pathogens” and “Non-pathogens” Groups V. fischeri ES114 V. fischeri MJ11 V. spendidus LGP32 V. cholerae O1 biovarE1 Tor N16961 V. cholerae O395 V. parahaemolytics RIMD_2210633 V. parahaemolytics BB220P V. vulnificus YJ016 V. vulnificus CMCP6 VV01 VV02 VV03 VV04 VV05 VN01 VN04 VN03 VN02 60 70 80 90 Similarity 100 Presence Absence A SIMPER test was performed to identify genes that lead to differences between Pathogens (V. cholerae, V. parahaemolyticus, V. vulnificus) and Non-Pathogens (V. fischeri, V. splendidus). Genes were supported by relative abundance in Pathogens, then by relative abundances in non-pathogens. Genomes are arranged based on the clustering pattern identified from the entire gene profile. Genes significantly enriched in a priori defined “Pathogens” and “Non-pathogens” Groups Transporters, transcription factors, hemolysins, exonucleases, carbohydrate metabolism (enormous gene variation) V. fischeri ES114 V. fischeri MJ11 V. spendidus LGP32 V. cholerae O1 biovarE1 Tor N16961 V. cholerae O395 V. parahaemolytics RIMD_2210633 V. parahaemolytics BB220P V. vulnificus YJ016 V. vulnificus CMCP6 VV01 VV02 VV03 VV04 VV05 VN01 VN04 VN03 VN02 60 70 80 90 Similarity 100 Presence Absence A SIMPER test was performed to identify genes that lead to differences between Pathogens (V. cholerae, V. parahaemolyticus, V. vulnificus) and Non-Pathogens (V. fischeri, V. splendidus). Genes were supported by relative abundance in Pathogens, then by relative abundances in non-pathogens. Genomes are arranged based on the clustering pattern identified from the entire gene profile. Genes significantly enriched in a priori defined “Pathogens” and “Non-pathogens” Groups V. fischeri ES114 V. fischeri MJ11 V. spendidus LGP32 V. cholerae O1 biovarE1 Tor N16961 V. cholerae O395 V. parahaemolytics RIMD_2210633 V. parahaemolytics BB220P V. vulnificus YJ016 V. vulnificus CMCP6 VV01 VV02 VV03 VV04 VV05 VN01 VN04 VN03 VN02 60 70 80 90 Similarity 100 Presence Absence A SIMPER test was performed to identify genes that lead to differences between Pathogens (V. cholerae, V. parahaemolyticus, V. vulnificus) and Non-Pathogens (V. fischeri, V. splendidus). Genes were supported by relative abundance in Pathogens, then by relative abundances in non-pathogens. Genomes are arranged based on the clustering pattern identified from the entire gene profile. A subset of Genes significantly enriched in a priori defined “Pathogens” and “Non-pathogens” Groups V. fischeri ES114 V. fischeri MJ11 V. spendidus LGP32 V. cholerae O1 biovarE1 Tor N16961 V. cholerae O395 V. parahaemolytics RIMD_2210633 V. parahaemolytics BB220P V. vulnificus YJ016 V. vulnificus CMCP6 VV01 VV02 VV03 VV04 VV05 VN01 VN04 VN03 VN02 60 70 80 90 Similarity 100 Presence Absence In yellow: Genes related to type 1 secretion, chemotaxis, permeases, proteases, and LPS synthesis (capsid polysaccharides, lipoproteins, exopolysacharrides) A subset of Genes significantly enriched in a priori defined “Pathogens” and “Non-pathogens” Groups V. fischeri ES114 V. fischeri MJ11 V. spendidus LGP32 V. cholerae O1 biovarE1 Tor N16961 V. cholerae O395 V. parahaemolytics RIMD_2210633 V. parahaemolytics BB220P V. vulnificus YJ016 V. vulnificus CMCP6 VV01 VV02 VV03 VV04 VV05 VN01 VN04 VN03 VN02 60 70 80 90 Similarity 100 Presence Absence Mostly hypotheticals (40), response regulators, glutathione synthase, starvation proteins A subset of Genes significantly enriched in a priori defined “Pathogens” and “Non-pathogens” Groups V. fischeri ES114 V. fischeri MJ11 V. spendidus LGP32 V. cholerae O1 biovarE1 Tor N16961 V. cholerae O395 V. parahaemolytics RIMD_2210633 V. parahaemolytics BB220P V. vulnificus YJ016 V. vulnificus CMCP6 VV01 VV02 VV03 VV04 VV05 VN01 VN04 VN03 VN02 60 70 80 90 Similarity 100 Presence Absence Hypotheticals (153), transcription factors (21), urease operon (10), lipoproteins (16), chemotaxis (8), zinc uptake (3), sideophore synthesis & uptake (6 – 2 operons), luciferase operon (3 genes) Genes significantly different between the Clinical and Environmental Strains of V. navarrensis V. fischeri ES114 V. fischeri MJ11 V. spendidus LGP32 V. cholerae O1 biovarE1 Tor N16961 V. cholerae O395 V. parahaemolytics RIMD_2210633 V. parahaemolytics BB220P V. vulnificus YJ016 V. vulnificus CMCP6 VV01 VV02 VV03 VV04 VV05 VN01 VN02 Drives separation VN03 VN04 60 70 80 90 Similarity 100 Endonucleases (5), Channel proteins (2), chemotaxis genes (5), permeases (2), transcriptional regulators (4), dehydratase (4) Hypotheticals, flagellar proteins Presence Absence 60 A Subset of Genes significantly different between the Clinical and Environmental Strains of V. navarrensis V. fischeri ES114 V. fischeri MJ11 V. spendidus LGP32 V. cholerae O1 biovarE1 Tor N16961 V. cholerae O395 V. parahaemolytics RIMD_2210633 V. parahaemolytics BB220P V. vulnificus YJ016 V. vulnificus CMCP6 VV01 VV02 VV03 VV04 VV05 VN01 VN02 VN03 VN04 70 80 90 Similarity 100 ATP dep. endonuclease Channel proteins Chemotaxis Endonucleases Phage tail collar domain Flagellin Transcript. regulators dehydratase Presence Absence Previously Discusses Virulence Factors Virulence Factor Description RTX Toxin rtxA gene encodes for the RTX toxin which is related with septicemia and gastroenteritis Hemolysins Exotoxins that lyse erythrocyte membranes by formation of pores with the liberation of iron binding proteins (transferrin, lactoferrin and hemoglobin). Four defined classes of Hemoylsins: TDH, TLH, δ – VPH, hlyA Experimental evidence suggests Hemolysins are involved in disease pathogenesis. Siderophores Low molecular weight compounds that have high affinity for iron molecules. Studies show the association of siderophores with virulence in Vibrios. Attachment Factors Toxin Co-regulated Pilus (TCP) and Type IV pilus Secretion Systems CTX is associated with Type II RTX is associated with Type I Capsular Polysaccharides •The most important virulence factor for V. vulnificus is its capsular polysaccharide (CPS). •V. vulnificus is an extracellular pathogen that relies on its CPS to avoid phagocytosis by host defense cells and complement (Linkous and Oliver, 1999; Strom and Paranjpye, 2000). •Unencapsulated mutants are susceptible to bactericidal activity in human serum (Shinoda et al., 1987). •Presence of capsule is related to the colony morphology (Yoshida et al., 1985; Wright et al., 1999). Class Capsular Polysaccharide Function Involved in subunit transport and flanked by direct repeat DNA sequence Gene wzb wzc LPS Biosynthesis Capsular polysaccharide biosynthesis Serum resistance genes Capsular polysaccharide biosynthesis Capsular polysaccharide biosynthesis system Serum resistance trkA V V V V V V V V V V V V V V N N N N 1 2 3 4 5 1 2 3 4 Selected Hemolysins Class Hemolysins Function HlyA (E1 Tor haemolysin) family Gene vvhA vvhB Similar to hemolysin III of B.sereus hlyIII Hemolysins vllY Virulence gene regulation hlyU V V V V V V V V V V V V V V N N N N 1 2 3 4 5 1 2 3 4 Iron Acquisition •Vibrio vulnificus pathogenecity - increased iron in the host results in increased susceptibility to infection (Weinberg 2000). •As with other invasive bacterial pathogens, iron-scavenging siderophores and proteins that bind host iron-containing proteins were identified in V. vulnificus. •A couple of studies indicated that the protease produced by V. vulnificus could be involved in acquisition of iron from heme proteins (Nishina et al., 1992; Okujo et al., 1996). •Litwin and Calderwood (1993) cloned the V. vulnificus fur gene, which encodes the central regulator in iron metabolism in many bacteria. •The essential role for vulnibactin in virulence was confirmed by Litwin et al. (1996). V. vulnificus mutant for vuuA, the ferric vulnibactin receptor, could not use vulnibactin and was decreased for virulence in mice. Class Iron acquisition Function Gene Central regulator in iron metabolism fur Ferric vulnibactin receptor vuuA Vulnibactin utilization protein viuB Siderophore synthase Vulnibactin synthase V V V V V V V V V V V V V V N N N N 1 2 3 4 5 1 2 3 4 Flagella and Motility Class Flagella and Motility Function Gene Encodes the flagellar basal body flgC Encodes flagellar hook protein flgE Involved in flagellar biosynthesis fliP V V V V V V V V V V V V V V N N N N 1 2 3 4 5 1 2 3 4 The mystery behind RTX toxin These following are the hits from the annotation for rtx: •RTX toxin – Toxin metabolic process; cytolysis •RTX protein – iron regulated protein When we BLAST these proteins with NCBI we found the following hits, •M6 family metalloprotease domain protein •Iron regulated protein frpC RTX machinery Class RTX Type 1 Secretion System Type IV Pilus Function Gene toxin rtxA ATP Binding cassette transporter for rtxA rtxB Essential acyclase of rtxA rtxC unknown function in transport rtxD Outer membrane protein tolC ABC transporter hlyB Membrane fusion protein hlyD Adherence (Present) V V 1 V V 2 V V 3 V V 4 V V 5 V N 1 V N 2 V N 3 V N 4 Some other interesting factors! Function Gene Heme receptor hupA DNA binding transcriptional regulator hupB Metalloprotease vvpE vvp15 Hypothetical protein Adherence to human epithelial cells vvp22 vvp28 pilD Relating to loss in cytotoxic activity purH Relating to decreased expression of Hemolysins toxR Autoinducer II production luxS pyrH toxS V V 1 V V 2 V V 3 V V 4 V V 5 V N 1 V N 2 V N 3 V N 4 Conclusions 1. V. navarrensis is unlikely to be a pathogen to healthy human individuals. • Absence of toxins • Absence of CPS • Presence of hemolysins similar to V. vulnificus 2. Very different profile from the compared Vibrios. 3. Vibrio navarrensis is not similar to the non-human pathogenic Vibrios. 4. Blood and environmental strains of V. navarrensis are very similar. • Differences: LPS synthesis, Type-I secretion system, Permeases. 5. We still believe that these will store a similar niche in the environment. 6. Vibrios are difficult to study owing to their metabolic versatility and wide range of animal hosts. 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